Chemiluminescence is a phenomenon where light is emitted from a chemical reaction that produces an excited-state product (Pinto da Silva, et al., (2012) Chemphyschem. 13: 2257). There is significant interest in developing chemiluminescent probes for use in point-of-care diagnostic devices, and for the imaging of biological activities in vitro and in vivo. Chemiluminescent probes can provide detection with high sensitivity and high contrast imaging due to exhibiting no initial background signal as a result of not requiring an external excitation source (Schuster et al., (1975) J. Am. Chem. Soc. 97: 7110). Moreover, chemiluminescent probes confer the additional advantages of not requiring an exogenous enhancer for activation or prior genetic modification for use.
Luminescent probes for use in detection of biological activities have been primarily based on four structures, an acridan ester, peroxalate, luminol, and luciferin platforms. Luminescent probes based on these platforms require in situ formation of an intermediate structure possessing a labile high-energy moiety (e.g., a dioxetanone) that induces the emission of light from the aromatic scaffold upon charge-transfer-induced decomposition (CTID) and subsequent relaxation to the ground state via a chemically-initiated electron exchange (CIEEL) mechanism.
The use of chemiluminescence in analyses has been primarily limited to luminol-based biochemical assays (e.g., Western blots) because the small quantity of light that is generated can be amplified through the use of chemical oxidants ([Ox]) and biological catalysts (enzymes), whereas all chemiluminescent probes based on other platforms are capable of generating only trace amounts of light in aqueous environments (with extremely short lifetimes) that are ill-suited for biological studies. Unfortunately, the enhancers (e.g., horseradish peroxidase) involved with catalyzing luminol-based reactions are typically exogenous to the cellular environment under study and must be administered at extremely high levels of concentration. On the other hand, bioluminescent probes (e.g., D-luciferin) do not require exogenous bioreagents for activation and emission of detectable amounts of light. However, bioluminescent probes do require an expressed enzyme (e.g., luciferase) from transfected genetic material in order to form a high-energy intermediate that is capable of emitting light upon activation, CTID, and CIEEL. As a result, bioluminescent probes are limited to xenograft models for in vivo studies.
A subclass of 1,2-dioxetanes that possess a protected meta-phenolate can be activated by a chemical event which releases a phenolate and triggers the production of light through CIEEL. The chemical event triggers the thermolysis of the 1,2-dioxetane moiety to generate a digroup intermediate that thereby, affords a high-energy species that emits light upon relaxation (electron exchange) to a stable ground state product. Sterically-hindered dioxetanes have been shown to possess greater thermal stability, hence the adamantane substituent (Schuster et al., (1975) J. Am. Chem. Soc. 97: 7110).
Furin is a serine endoprotease that is responsible for proteolytic processing in the body (Thomas, G. (2002) Nature Revs. Mol Cell Biol. 3: 753). Furin is a ubiquitous enzyme that is upregulated in response to various environmental conditions, such as hypoxia and cytokine stimulation, which are characteristics of human tumors (Bourne & Grainger (2011) J. Immunol. Methods 364: 101). It has been shown to be upregulated in several cancers including glioblastomas and is directly correlated with increased cancer aggressiveness due to its role in degredation of the extracellular matrix (ECM) that promotes intravasation and metastasis of tumor cells (Thomas, G. (2002) Nature Revs. Mol Cell Biol. 3: 753). Specifically, furin cleaves C-terminal to basic residues and is specific for the peptide sequence R-X-R/K-R (Arg-X-Arg/Lys-Arg), where “X” is variable (most commonly valine). This sequence can be integrated into a probe that contains an optical reporter in order to monitor both the location and degree of enzyme activity within a specimen. Such “smart” probes have been used in biological systems for monitoring enzymatic activity with great success (Dragulescu-Andrasi et al., (2013) J. Am. Chem. Soc. 135: 11015; Madan et al., 2013) PloS one 8: e79065; Huang et al., (2102) Bioconjugate Chem. 23: 2159; Edgington et al., (2011) Curr. Opinion Chem. Biol. 15: 798).
Despite extensive development of chemiluminescent probes, there remains a long-felt need for fluorophore-based probes that offer advantages over existing probes. For example, greater levels of chemiluminescent light intensity that can be detected in animal tissues at depth within a body is an existing deficiency in the repertoire of available probes.